Interaction of the MCC with the APC/C
Regulation and tight control of mitotic progression is essential for eukaryotic cell proliferation. One major regulator of cell division is the anaphase promoting complex/cyclosome (APC/C); failure of the APC/C causes improper sister chromatid separation. The APC/C is a stable multiprotein complex consisting of at least 12 known core subunits. The core subunits bind the adaptor proteins Cdc20 for mitotic or Cdh1 for interphase regulatory function. APC/C activity is regulated by a number of spindle proteins. This protein network forms the spindle checkpoint, a surveillance mechanism that delays anaphase onset until all chromosomes are correctly attached to the mitotic spindle in a bipolar fashion. Until proper attachment of the microtubuli to the outer kinetochores, the MCC (mitotic checkpoint complex) is formed inactivating the APC/C. This changes when the last kinetochore becomes attached to microtubuli. Now, Cdc20 activates the APC/C which in turn ubiquitinylates the cohesin inhibitor securin. This leads to cohesin degradation, initiating sister chromatide separation. In early G1, instead of Cdc20, Cdh1 activates the APC/C to maintain mitotic cyclin destruction.
Although the architecture of the human APC/C is well described, knowledge on the interaction between APC/C and MCC is limited. Also little is known about the time resolved localization of the proteins and complexes. We plan to define the interaction partners within the complexes and study their dynamics and molecular interactions by fluorescence microscopy in living cells by FRAP, FRET, FCS, and FLIM. The molecular interactions will also be studied in vitro by Yeast-Two-Hybrid analysis, among themselves and with all proteins of the kinetochore. Cloning of these proteins is in progress.
Although the architecture of the human APC/C is well described, knowledge on the interaction between APC/C and MCC is limited. Also little is known about the time resolved localization of the proteins and complexes. We plan to define the interaction partners within the complexes and study their dynamics and molecular interactions by fluorescence microscopy in living cells by FRAP, FRET, FCS, and FLIM. The molecular interactions will also be studied in vitro by Yeast-Two-Hybrid analysis, among themselves and with all proteins of the kinetochore. Cloning of these proteins is in progress.
